Quantitative determination of uric acid

ABSTRACT

Uric acid is determined by contacting a sample suspected of containing uric acid with a phosphate buffer, an NADH solution, ethanol, alcohol dehydrogenase and catalase in the presence of air to form a reaction mixture containing 0.3 to 0.7 volume percent alcohol, measuring a first extinction value, adding uricase to the reaction mixture, measuring a second extinction value spectrophotometrically and taking the difference between the extinction values as a measure of the initial uric acid content in the sample.

United States Patent Stork [4 Jan. 21, 1975 [75] Inventor: Harald Stork,

Mannheim-Feudenheim, Germany [73] Assignee: Boehringer Mannheim QII'IIZH,

Mannheim, Germany [22] Filed: July 17, 1973 [21] Appl. No.: 380,020

[30] Foreign Application Priority Data Aug. 2, 1972 Germany 22379404 May 25, 1973 Germany 23267563 [52] US. Cl 195/1035 R [51] Int. Cl Cl2k l/00, C07g 7/02 [58] Field of Search 195/1035 R [56] References Cited UNITED STATES PATENTS 3,335,069 8/1967 Philip 195/1035 R 3,349,006 10/1967 Gregory l95/l03.5R

OTHER PUBLICATIONS Kageyama, N., Colorimetric Determination of Uric Acid using Ulicase-Catalase, Chemical Abstracts, Vol. 71, 1969 (p. 49).

Primary Examiner-David M. Naff Attorney, Agent, or FirmBurgess, Dinklage & Sprung [57] ABSTRACT Uric acid is determined by contacting a sample suspected of containing uric acid with a phosphate buffer, an NADH solution, ethanol, alcohol dehydrogenase and catalase in the presence of air to form a reaction mixture containing 0.3 to 0.7 volume percent alcohol, measuring a first extinction value, adding uricase to the reaction mixture, measuring a second extinction value spectrophotometrically and taking the difference between the extinction values as a measure of the initial uric acid content in the sample.

10 Claims, 2 Drawing Figures PATENTEBJAN 21 I975 FIG. I.

CALIBRATION CURVE WITH AQUEOUS URIC .ACID

6 I URIC ACID CONCENTRATION 9/lO0rr DEPENDENCE OF EXTINCTION ON THE I 0.05 O.I v "II-SERUM (URIC ACID CONTENT ssmg/Ioowa QUANTITATIVE DETERMINATION OF URIC ACID The present invention is concerned with a method for the quantitative photometric determination of uric acid. More specifically, the invention relates to such a determination which can be carried out completely enzymatically, the results obtained being such that they can be read off in the near ultra-violet range of a photometer.

Hitherto, in principle, two methods have been known for the determination of uric acid.

In one case, uric acid is oxidized by phosphotungstic acid and the phosphotungsten blue formed by the reaction is measured colorimetrically. Other reducing substances, for example, medicaments or reducing metabolites, can, of course, have a disturbing effect on the reaction.

ln the second case, which is the uric acid determination by Praetorius method, uric acid is decomposed by means of uricase to give allantoin, the absorption decrease of the uric acid in the region of 293 mp. being a measure for its concentration. This process is, like most enzyme reactions, very specific but the inherent absorption of the protein component ofthe enzyme has a disturbing effect, in the same way as other proteinaceous materials present in the sample, so that false results can be obtained.

In order to avoid the above-mentioned disadvantages, it has been suggested to determine the hydrogen peroxide, colorimetrically formed by the enzymatic reaction of uric acid with uricase, (see Bernt and Lorenz, Anal. Biochem., 1969). The determination depends upon the oxidation of o-dianisidine by means of peroxidase to give a colored material.

Although the extinction can thereby be measured the most favorably in the visible range of the spectrum, the process suffers from a number of disadvantages which make it unsuitable for practical purposes. On the one hand, the use of o-dianisidine is nowadays avoided because of the danger of a cancerogenic effect and, on the other hand, this process is also disturbed by proteins so that deproteinisation is essential before carrying out the uric acid determination.

Furthermore, German Pat. No. 2,149,675 describes a process in which methanol is oxidized to formaldehyde by the enzymatically-formed hydrogen peroxide in the presence of catalase and the formaldehyde then reacts, in the presence of an ammonium salt according to Hantzschs reaction, with acetylacetone to give 3,5- diacetyl-l ,4-dihydrolutidine. The concentration of this yellow-colored material, which is proportional to the concentration of the uric acid, can be determined colorimetrically at 410 mu. This process is certainly an improvement over the previously known methods of determination but it is laborious because of the neces sity of having to maintain certain reaction parameters. Thus, for example, a bath thermostatically controlled at 37C. is necessary in which, for the development of the colored lutidine compound, it is necessary to incubate the test sample for at least 30 minutes but, practically speaking, for more than an hour. Furthermore, the last stage of the process does not take place enzymatically and can, therefore, be disturbed by reducing or oxidizing components present in the sample, which react with the formaldehyde.

The present invention provides a method for the detection of uric acid which can be carried out completely enzymatically and which, in a short period of time, gives, without laborious incubation, a quantitative result which is not influenced by substances which disturb the previously known methods for determining uric acid.

Essentially, the method of the invention comprises reacting the hydrogen peroxide liberated by the allantoin reaction of uric acid with ethanol in the presence of catalase, reducing the acetaldehyde thereby formed back again to ethanol by reduced nicotinamide-adenine-dinucleotide (NADH) in the presence of alcohol dehydrogenase, and measuring the decrease of the NADH concentration spectrophotometrically in the favorable range of 340 or 366 my, as being proportional to the uric acid content of the sample.

The individual steps involved in this method may be old per se but they have previously not been combined because it was assumed that the reactions would mutually disturb one another. For a better understanding of the course of the reactions involved, they are illustrated by the following equations:

Reaction A:

Reaction B:

Reaction C:

ld hy H+ alculml IIIZLUIHIIIHIIIIXW etha nol NAD It is known that the three stages proceed at different optimum pH values, i.e., (A) at pH 9.3, (B) at pH 7.0 and (C) at pH 6.5. The relatively alkaline pH optimum of the uricase reaction (A) is especially critical, having regard to the known very pH-dependent equilibrium adjustment of reaction (C).

it is also known that reaction (C) is disturbed by ethanol present in the sample. Furthermore, it is known that catalase is not an exclusively peroxidate-acting enzyme but that it, especially in the case of small concentrations of ethanol, preponderantly liberates oxygen directly from hydrogen peroxide which, as such, is not able to react with ethanol.

Since, however, reaction (B) requires, for a quantitative reaction of the hydrogen peroxide, a high ethanol concentration, whereas ethanol has an inhibiting action on the course of reaction (C) (cf. Methoden der enzymatischen Analyse, H. U. Bergmeyer, I962, p. 292), it is most surprising that critical conditions could be found which enable the process steps B and C to be combined without substantially disturbing the quantitative course of the reaction as a whole. By the additional combination of steps B and C with process step A, the relationships involved become, of course, even more complex.

Thus, according to the present invention, there is provided a process for the quantitative determination of uric acid, wherein uricase and catalase act upon a uric acid-containing sample in the presence of ethanol and of air to form acetaldehyde, which is reduced back again to ethanol by the action of alcohol dehydrogenase with NADH, the decrease of the NADH concentration being measured spectrophotometrically, the process being-carried out at a pH of 6.3 to 7.1 and such a quantity of ethanol is added to the reaction mixture that the concentration thereof is within the limits of 0.3 to 0.7 vol.%.

Good results are obtained when operating at a pH of 6.3 to 6.8 or at a pH of about 7.0.

allantoin H 0 CO By maintaining the above-described conditions, it is possible, surprisingly, to combine process steps A, B and C. Outstandingly correlated results are obtained and the reaction is not noticeably disturbed by protein or by foreign materials. Since the acetaldehyde formed is also immediately reduced by NADH at ambient temperature and is thus removed from the equilibrium. the reaction is also relatively insensitive to disturbing reducing substances.

The optimum ethanol concentration for the reaction is about 0.5 vol.'/1.

As buffer for the maintenance of the essential pH range. especially favorable results have been obtained with the use of a phosphate buffer containing potassium dihydrogen phosphate and disodium hydrogen phosphate. Both compounds, when mixed in an appropriate ratio, give the required pH value between 6.3 and 7.1. Thus, for example, a mixture of 3.522 g. potassium dihydrogen phosphate and 7.268 g. disodium hydrogen phosphate, dissolved in water, buffer at pH 7.

Although. in the case of conventional acetaldehyde determinations, process step C is normally only carried out after a deproteinization of the sample. we have, unexpectedly, found that. in the case of the process according to the present invention. deproteinizing is unnecessary (see also FIG. 11 of the accompanying drawings).

The extinction of the NADH can be measured in the near ultra-violet range at 340 or 366 mu.

The process according to the present invention can be carried out at ambient temperature. a thermostatic control being unnecessary. The analysis results are obtained after about 20 minutes.

In comparison with the simple uric acid determination by Praetorius method, the results obtained must admittedly be provided with an additional correction factor. Since. however, this is a constant average difference, the exactitude of the determination is scarcely impaired.

The process according to the present invention only requires a very small amount of sample (0.1 ml. of serum) so that it can also be used for carrying out determinations with capillary plasma.

The invention is also illustrated in the accompanying drawings in which:

HO. 1 represents a calibration curve produced with an aqueous solution of uric acid.

FlG. 11 is a calibration curve which has been determined by means of a standard serum with a predetermined content of 5.9 mg.72 uric acid.

From FlGS. I and 11, there can be seen the linearity of the relationship between the extinction and uric acid concentration.

In the following Tables 1 and 2. a comparison is made between the uric acid values obtained from human serum by means ofthe process according to the present invention and by absorption measurement using Praetorius method.

The following Examples are given for the purpose of illustrating, without limitation, the present invention:

EXAMPLE 1.

a. Reagents and reagent solutions 1. Phosphate buffer: 3.522 g. potassium dihydrogen phosphate and 7.268 g. disodium hydrogen phosphate dihydrate were dissolved in 1,000 ml. double distilled water.

2. NADH solution: 25 mg. NADH were dissolved in 10 ml. of a 1% aqueous sodium bicarbonate solution. This solution is stable for about 10 days.

3. Ethanol (absolute).

4. Alcohol dehydrogenase (Boehringer Mannheim, Germany): crystal suspension in 3.2 M ammonium sulphate solution. pH about 6; specific activity 200 U/mg. (25C.).

5. Catalase (Boehringer Mannheim, Germany): solution in 3071 glycerol/10% ethanol; specific activity 260,000 U/ml. (25C.).

6. Uricase (Boehringer Mannheim, Germany): solution in 50% glycerol. pH 10.2 50 mM glycine, 0.13 M sodium carbonate; specific acivity 4.5 U/mg. (25C.).

7. Uric acid standard solution (5 mg./l00 ml.): 5 mg. uric acid were dissolved with 0.1N aqueous sodium hydroxide solution and made up to ml.

8. Uric acid standard serum (5.9 mg./100 ml. there was used a standard serum from the firm Myegaard (available under the Registered Trade Mark Seronorm").

b. Carrying out of the uric acid determination Into a 1 cm. cuvette were successively pipetted 2.0

ml. phosphate buffer l 0.15 ml. NADH solution (2 0.01 ml. ethanol (3), 0.01 ml. alcohol dehydrogenase (4), 0.005 ml. catalase (5) and. finally, 0.] ml. of the sample was tested. Mixing was briefly carried out after each pipetting and the first extinction (E,) was read off at 366 mu 10 minutes after the addition of the sample. Immediately after reading off E 10 a1. uricase (6) was pipetted in. After a further 10 minutes, the second extinction value (E was read off at the same wavelength. The blank value (L) was read off against water.

The uric acid content was calculated as follows:

A E X 131 mg.7z uric acid The factor of 131 was determined empirically (cf. Table 2). Comparative experiments 1. Groups each of 10 samples were determined in parallel by means of the process according to the present invention and according to Praetorius method. The results expressed as mgf71. uric acid, are shown in the following Table 1:

TABLE 1 Uric acid detennination of 10 samples A: by Praetorius' method (1 cm. cuvette, 293 m p.) B: by the process according to the present invention (1 cm. cuvette, 366 m p).

From Table 1, there can be calculated statistically, for the process according to the present invention, a variation coefficient of 2.0% whereas from the Praetorius method, there is obtained a coefficient of 1.8671. 2. 32 different human serum samples with different uric acid contents were determined in parallel by means of the process according to the present invention and by Praetorius method. In the following Table 2, there are set out the results obtained, from which was obtained a percentage error spread of the deviations to be observed of 12.6 to l4.06/1.. From this is calculated the factor 131 (average error 13% The calculation of the linear regression accordingly gave a coefficient of l 0.99.

TABLE 2 Uric Acid determination in human serum results of the results of the cal graphic evaluculation with the ation by the factor l3l according results of thc determination by Praetorius method process of the to the process of the present invention present invention (mgf/l) (mgf/r) (mg/71) EXAMPLE 2.

6. Uricase (Boehringer Mannheim, Germany): solution in 50% glycerol pH 10.2 50 mM glycine; 0.13M sodium carbonate; specific activity 4.5 U/mg. (C.).

7. Uric acid standard solution (12 mg./l00 ml): l2

mg. uric acid were dissolved in 0.lN aqueous sodium hydroxide solution and made up to I00 ml.

b. Carrying out of the uric acid determination.

This was carried out in a manner analogous to that described in Example lb.

5 Samples ofuric acid standard solution (cf. Example 2a, 7) were determined in parallel by the process according to the present invention and by Praetorius' method. The following values (mg/100 ml.) were obtained by means ofthe process according to the present invention: 12.2; l2.2; 12.2; H6 and ll.5.

As can be seen, on average 100% uric acid values were found.

EXAMPLE 3.

The reagents used in Examples 1 and 2 can advantageously also be mixed for one or more tests in the volume ratios set out in section b). e

The test volume taken from the mixture can then. in a single further pipetting step, be mixed with the sample to be tested (serum or urine or other uric acidcontaining solution) so that the laborious individual pipettings can be omitted. This reagent mixture is of limited stability.

It will be understood that the foregoing specification and examples are illustrative but not limitative of the present invention inasmuch as other embodiments within the spirit and scope of the invention will'suggest themselves to those skilled in the art.

What is claimed is:

1. Process for the quantitative determination of uric acid in a sample suspected of containing same, which process comprises contacting said sample with a phosphate buffer, an NADH solution, ethanol, alcoholdehydrogenase, and catalase in the presence of air, wherein the ethanol concentration is from 0.3 to 0.7 volume percent of the resulting reaction mixture, whereby acetaldehyde is formed, and measuring a first extinction value, thereafter contacting the reaction mixture with uricase and measuring a second extinction value spectrophotometrically at 340 or 366 mu, and taking the difference between the extinction values as a measure ofthe initial uric acid content in said sample, wherein the process steps are carried out at a pH of from 6.3 to 7.l'.

2. Process as claimed in claim 1, wherein said pH is about 7.0.

3. Method as claimed in claim 1, wherein said pH is in the range from 6.3 to 6.8.

4. Method as claimed in claim I, wherein said ethanol concentration is about 0.5 volume '/I-.

5. Method as claimed in claim I, wherein the method is carried out at ambient temperature.

6. Method as claimed' in claim I, wherein said pH is maintained by the use of a buffer containing potassium dihydrogen phosphate and disodium hydrogen phosphate.

7. Method as claimed in claim I, wherein said NADH solution is supplied in the form of aqueous solution containing about l'/r sodium bicarbonate.

10. Method as claimed in claim 1, wherein said uricase is supplied in the form of a solution in 50% glycerol, pH 10.2 50 mM glycine, (H3 M sodium carbon- 

2. Process as claimed in claim 1, wherein said pH is about 7.0.
 3. Method as claimed in claim 1, wherein said pH is in the range from 6.3 to 6.8.
 4. Method as claimed in claim 1, wherein said ethanol concentration is about 0.5 volume %.
 5. Method as claimed in claim 1, wherein the method is carried out at ambient temperature.
 6. Method as claimed in claim 1, wherein said pH is maintained by the use of a buffer containing potassium dihydrogen phosphate and disodium hydrogen phosphAte.
 7. Method as claimed in claim 1, wherein said NADH solution is supplied in the form of aqueous solution containing about 1% sodium bicarbonate.
 8. Method as claimed in claim 1, wherein said alcohol dehydrogenase is supplied in the form of a crystal suspension in 3.2 molar ammonium sulfate solution.
 9. Method as claimed in claim 1, wherein the said catalase is supplied in a solution of 30% glycerol/10% ethanol.
 10. Method as claimed in claim 1, wherein said uricase is supplied in the form of a solution in 50% glycerol, pH 10.2 50 mM glycine, 0.13 M sodium carbonate. 